SUMMARY

Some snakes have a feeding regime characterized by the infrequent ingestion
of relatively large meals, causing impressive increments in post-prandial
metabolism. Metabolism remains elevated for many days, while digestion
proceeds, resulting in considerable investment of time and energy. Snakes
actively adjust thermoregulatory behavior to raise their body temperature
during digestion, exhibiting a post-prandial thermophilic response that
accelerates digestion at the expense of higher metabolic rates. In the present
study, we investigated the possibility that endogenously derived heat,
originating as a byproduct of the post-prandial increase in metabolism, could
itself contribute to the elevated body temperature during digestion in the
South American rattlesnake Crotalus durissus. We assessed heat
production, at a constant environmental temperature, by taking infrared (IR)
images of snakes during fasting and after being fed meals varying from 10% to
50% of their own body masses. Our results show clearly that digesting
rattlesnakes have significantly increased body temperatures, even when
precluded from adjusting their thermoregulatory behavior. The feeding-derived
thermogenesis caused the surface body temperature of rattlesnakes to increase
by 0.9–1.2°C, a temperature change that will significantly affect
digestive performance. The alterations in body temperature following feeding
correlated closely with the temporal profile of changes in post-prandial
metabolism. Moreover, the magnitude of the thermogenesis was greater for
snakes fed large meals, as was the corresponding metabolic response. Since IR
imaging only assesses surface temperatures, the magnitude of the thermogenesis
and the changes in deep core temperature could be even more pronounced than is
reported here.

As in other ectothermic organisms, temperature exerts a pervasive influence
on all activities in snakes. Different activities, however, are affected
differently by changes in body temperature
(Stevenson et al., 1985;
Van Damme et al., 1991), and
thus different activities (e.g. locomotion, digestion) may be optimal at
different temperatures. As a consequence, snakes may alter their body
temperature in activity specific patterns. For example, if given a choice of
environmental temperatures during digestion, snakes
(Cowles and Bogert, 1944;
Regal, 1966;
Walker and Taylor, 1966;
Greenwald and Kanter, 1979;
Huey, 1982; Slip and Shine,
1988a,b;
Jaeger and Gabor, 1993;
Sievert and Andreadis, 1999),
as well as other reptiles (see Huey,
1982), behaviorally increase their preferred body temperature, the
so-called post-prandial thermophilic response. The primary consequence of such
an increase in body temperature, in almost all cases examined, is a shortening
of digestion time at the expense of increased rates of metabolism during the
period of SDA (Wang et al.,
2003; Toledo et al.,
2003). Toledo et al.
(2003), however, found that
the SDA of boas fed different meal sizes was energetically less costly at
30°C than at 25°C. Thus, at least in this one particular case, it
seems that the post-prandial thermophilic response was advantageous not only
by decreasing the duration of digestion but also by improving the energetic
return on the meal.

The post-prandial thermophilic response has always been associated with
adjustments in thermoregulatory behavior that would allow animals to alter
body temperature by exploring the natural heat sources available in their
environment (e.g. Blouin-Demers and
Weatherhead, 2001). The possibility of using metabolism as a
source of heat to increase body temperature was thought to be of minor
importance, perhaps due to the widespread belief that ectothermic vertebrates
in general (Pough, 1983), and
some snakes in particular, have a relatively low aerobic capacity
(Ruben, 1976;
Lillywhite and Smits, 1992),
that would preclude any considerable heat generation by metabolic means. Such
an interpretation, although generally true, ignores the fact that the
metabolism of some snakes may increase by up to 17-fold during digestion
(Secor and Diamond, 1995,
2000), which will generate a
considerable amount of heat (Benedict,
1932; Van Mierop and Barnard,
1976; Marcellini and Peters,
1982). Therefore, in the present study, we investigated whether
snakes could use this heat source. Specifically we examined whether the body
temperature of the South American rattlesnake Crotalus durissus
terrificus is affected by the increased metabolic rate experienced during
digestion at a constant environmental temperature using infra-red (IR) imaging
technology. We took IR pictures from digesting and non-digesting snakes for a
period of 7 days, and compared their body surface temperature with the ambient
temperature. We also examined the effects of meal size on the magnitude of the
thermogenesis in these snakes, since meal size is one of the most influential
determinants of the SDA response: the larger the meal, the greater the
post-prandial metabolism and the longer the duration of the SDA
(Andrade et al., 1997;
Toledo et al., 2003). We
hypothesized that body temperature following feeding (i.e. thermogenesis)
would rise with a similar time course to the SDA that is well characterized in
snakes, and that the magnitude and duration of the metabolic, post-prandial
thermogenesis would be positively correlated with meal size.

Materials and methods

Animals

Juvenile rattlesnakes Crotalus durissus L. were born in captivity
from females collected in São Paulo state, southeastern Brazil. They
were housed in individual wood cages (25 cm×26 cm×26 cm) with
lateral holes for ventilation and a glass-front sliding door in a temperature
controlled room (30±2°C). Snakes were fed every other week with
mice and rats (meal mass approximately 30% of snake body mass
Mb) and were given free access to water. In total, 18
animals were used in this study (mean Mb 341±21 g).
Animals were fasted for 2 weeks before experimentation, and only individuals
that seemed healthy and that were not moulting were used.

Experimental protocol

Three groups of snakes were used in this study. The control group
(N=4) was fasted throughout all measurements. The remaining snakes
were fed varying numbers of mice (either 1, 2 or 3) to create a small meal
group and a large meal group. The small meal group (N=6) was fed
enough mice to produce a meal ranging from 10–25% (average 19.4%) of the
snake's pre-meal Mb. The large meal group (N=8)
was fed enough mice to produce a meal ranging from 26–50% (average
33.7%) of the snake's pre-meal Mb (see
Table 1 for details of snake
body masses and meal masses). These mice were consumed within 30 min of
presentation to the snakes. Prior to feeding, an IR thermal image was taken of
each snake, serving as the fasted (time 0) value. Immediately following this,
snakes were either allowed to continue to fast, or fed the appropriate number
of mice, and thermal images were taken again 3, 16, 24, 36, 48, 64, 72, 96,
120, 144 and 168 h later. These images allowed the precise determination of
both snake surface temperature and local ambient temperature within the
individual animal's cage.

Infrared imaging

IR thermal images were taken with a MikroScan 7515 Thermal Imager (Mikron
Infrared®, Oakland, NY, USA). This device produces a 12-bit image
(320×240 pixels) and stores the temperature information of each pixel at
a resolution of 0.1°C. All temperature readings are automatically
corrected for nonblackbody properties by assuming an emissivity of 0.95, which
is a reasonable estimate for biological tissues. We assessed the validity of
this assumption by examining the IR from black electrical tape (known
emissivity = 0.95) held at the same temperature as snake skin. Both the tape
and the skin were found to radiate the same degree of IR, suggesting that an
emissivity of 0.95 is a safe assumption. The procedure for taking IR images
involved briefly opening the glass front of the cage and taking an image at a
distance of 30–45 cm from the snake. The snakes were well accustomed to
this procedure by the time the experiment started, and most individuals simply
remained coiled and passive as the image was taken. No changes in body surface
temperatures associated with agitation were observed.

Data analysis and statistics

IR images were analyzed using MikroSpec RT (Mikron Infrared®) software.
Regions of interest on the body were outlined and the average surface
temperature determined. Since there was little variability in the surface
temperature of the body (except for the head region), random regions
(comprising approximately 10% of the body surface area) were used to determine
body surface temperature (hereafter referred to as body temperature,
Tb). The background temperature of the wooden cage was
also determined, and served as a local ambient temperature comparison. The
difference between body temperature and ambient temperature
(ΔT) was determined for every snake at every time point. To aid
in the analysis, the maximum ΔT during SDA, the time at which
the maximum ΔT occurred, and the area under theΔ
T curve during SDA were determined for each individual snake.
A one-way ANOVA using a Bonferroni post-hoc comparison was used to
test the significance of all changes in these three variables (max.Δ
T, time of max. ΔT, and area under theΔ
T curve). The comparison between pre- and post-feeding values
of ΔT were made using a one-way repeated measures analysis of
variance (ANOVA), followed by the post-hoc Dunnett's test, which
tested for differences between post-feeding values against a control value
(pre-feeding value). Differences were considered significant when
P<0.05.

Results

Meal size

Feeding variables are summarized in
Table 1. There were no
significant differences between the masses of snakes used in each group.

Thermal increment of feeding

Fasted snakes did not show any significant changes in ΔT
throughout their fasting period (Fig.
1), although ΔT did fluctuate over time by
approximately –0.1°C to +0.1°C.

The difference between body surface temperature and the ambient temperature
(ΔT) during the 168 h following feeding. Filled circles
represent the fasted group of snakes, open circles represent the snakes fed a
small meal (10–25%Mb), and the filled triangles
represent the snakes fed a large meal (26–50%Mb).
*Significant difference between the small meal value and its
pre-feeding value (time 0); †significant difference between
the large meal value and the pre-feeding value (time 0). The dotted line
represents the oxygen uptake for snakes fed a diet of
20%Mb, and the broken line represents the oxygen uptake
for snakes fed a diet of 30%Mb (taken from
Andrade et al., 1997).

Snakes fed both small and large meals demonstrated significant and
sustained increases in Tb following feeding
(Table 2; Figs
1,
2). Within 3 h of being fed a
meal, both the small and the large meal group exhibited a significant rise inΔ
T, which remained above the pre-feeding value for up to 6 days
(144 h; Fig. 1). The maximumΔ
T of 0.93±0.11°C in the small meal group occurred
on average 23±4 h post-feeding, whereas the maximum ΔT
of 1.3±0.04°C in the large meal group occurred 42±7 h
post-feeding. Both the maximum ΔT and the time at which maximumΔ
T occurred were significantly higher in the large meal than in
the small meal group. Furthermore, the total area under the ΔT
curve during the SDA period was significantly higher in the large meal group
than in the small meal group (Table
2). Interestingly, the rate of rise of ΔT was
identical in both groups. Slight regional variations in surface temperature
did exist in some snakes (Fig.
2),however, most surface temperatures over most of the snake's
body were relatively uniform (<0.2°C difference).

Infrared thermal image of a rattlesnake (A) prior to feeding and (B) 48 h
following feeding a meal comprising 32% Mb. The scale bar
shows a total range of 2.5°C, where black is the coldest temperature and
white is the warmest temperature. Note the uniform increase in body surface
temperature in the snake following feeding. The darkest spot in each image is
the nose, where evaporative cooling leads to a significant reduction in
temperature.

The effect of diet size is further demonstrated in the correlations between
maximum ΔT and the SDA area versus meal size for
individual animals (Fig. 3).
Significant positive correlations exist between maximum ΔT and
meal size (%Mb) (r2=0.83) and between
SDA area and meal size (%Mb)
(r2=0.83), suggesting tight correlations between these
variables, although there was a tendency for the relationships to asymptote at
the largest meal sizes, suggesting that diet-induced thermogenesis does not
continue to increase linearly with meal size.

(A) Individual values for the peak thermal increment (ΔT)
following feeding at different meal sizes as a function of snake body mass.
(B) Individual values for the total area under the SDA temperature curve
following feeding at different meal sizes. Pearson's coefficients are shown in
both graphs.

Discussion

Critique of methods

Although IR thermography has not seen widespread use in physiology, its
non-invasive nature makes it an ideal method for rapidly assessing multiple
surface temperatures in a large number of animals. The technology has reached
the stage where resolution and the accuracy rival that of other temperature
recording devices. The largest error in using this technique occurs in knowing
the emissivity of the target. Most biological tissues exhibit an emissivity of
0.95, which implies that they emit 95% of the radiation emitted by an ideal
blackbody radiator at the same temperature
(Speakman and Ward, 1998). The
emissivity of snake skin is unknown; however, when IR image comparisons were
made between the snakes' surface temperatures and the surface temperature of a
substance of known emissivity, there were no discernable temperature
differences, inferring that we have used a valid emissivity correction factor
in the determination of surface temperatures.

Specific dynamic action and meal size effects on thermogenesis

By overlapping the thermal increment associated with feeding (present
study) with the post-prandial metabolic response of rattlesnakes
(Andrade et al., 1997), a clear
correlation emerges between both variables (See
Fig. 2). While digesting meal
sizes 10–50% of their own body masses, this species experiences peaks in
metabolism between 15 h and 33 h post-feeding, at values 3.7- to 7.3-fold
higher than the values measured during fasting
(Andrade et al., 1997).
Similarly, we have found that thermogenesis attained greater magnitude in
those snakes fed with larger meals and that the attainment of peak values in
Tb occur in accordance with the peak in metabolism. It
thus appears that the thermal effect of feeding that we recorded reflects a
total body temperature increment arising from the SDA, as previously
conjectured by Benedict
(1932).

There are other possible explanations for the source of this heat
production. Marcellini and Peters
(1982) conjectured that
undetectable muscular contractions and chemical decomposition of food may have
contributed substantially to the post-prandial thermogenesis of snakes. Our
data, however, suggest that the latter is unlikely. Indeed, we observed that a
decaying, uneaten mouse produced no significant heat under the same
experimental conditions (G. J. Tattersall, unpublished data). Further, the
only increase in muscular activity that could be anticipated for digesting
snakes is an increase in gut motility, since activity in general is decreased
in fed snakes (Beck, 1996).
This renders it improbable that an undetectable increase in muscular activity
might have been involved in the increase in heat production after feeding. The
maintenance of all snakes in a temperature-controlled room, with no
possibility of changing heat exchange rates by behavioral means, excludes the
possibility that the increment in body temperature exhibited by fed
rattlesnakes is the result of an adjustment in thermoregulatory behavior, i.e.
a post-prandial thermophilic response. Finally, the rattlesnakes' body
temperatures returned to fasting levels with a time course that is in good
agreement with the duration of the metabolic SDA response recorded for this
species (Andrade et al.,
1997).

For rattlesnakes, our results suggest that all beneficial consequences
associated with the post-prandial thermophilic response listed above may be
achieved not only by altering thermoregulatory behavior, but also through the
thermogenic consequences of the elevated metabolism during digestion. In
C. durissus, we have found that thermogenesis alone may account, on
average, for a 0.9–1.2°C increase in body temperature during the
first 2–3 days after feeding. The important question is whether such an
increase would be of any physiological significance to the rattlesnake's
digestion. We tried to address this issue by calculating the effect of a
1°C change in body temperature on the digestion of snakes, by regressing
SDA duration and SDA cost (expressed as a percentage of the calorific content
of the meal, i.e. SDA coefficient; see
Toledo et al., 2003) against
body temperature, using a set of data obtained for C. durissus at
25° and 30°C (S. P. Brito, A. S. Abe and D. V. Andrade, unpublished
data). This procedure revealed that a 1°C increase in body temperature,
under the conditions in which we performed the experiments, may account for a
19 h decrease in SDA duration and a 0.3% decrease in the SDA coefficient.
Thus, the thermogenic effect of feeding, per se, may, indeed, affect
the digestive performance and the duration of digestion in rattlesnakes.
Moreover, the ability to increase body temperature after feeding by
thermoregulatory behaviors is reported to be constrained in rattlesnakes by
the availability of adequate thermal microhabitats, reduced mobility and
reclusive behaviors (Beck,
1996). Thus, it seems possible that the beneficial effects of
metabolic thermogenesis on digestion may assume a greater importance during
the night, on cloudy days, or whenever behavioral thermoregulation and the
achievement of the post-prandial thermophily are constrained. Finally, by
using the infrared imaging technique, we assessed only body surface
temperature and, therefore, differences in deep core body temperature due to
digestion associated thermogenesis may be even larger. Indeed, in experiments
performed with pythons fed with meals containing temperature data loggers,
Marcellini and Peters (1982)
were able to detect increases in body temperature up to 4°C (see also
Benedict, 1932;
Van Mierop and Barnard, 1976).
Moreover, digesting pythons experience metabolic responses that are far larger
than those observed in rattlesnakes
(Andrade et al., 1997;
Secor and Diamond, 2000),
which could also contribute to the larger thermogenic effect of feeding
exhibited by this species (Benedict,
1932; Van Mierop and Barnard,
1976; Marcellini and Peters,
1982).

The thermogenic effect of feeding has been examined in one lizard species
by Bennett et al. (2000) who
found that digesting Varanus at 32 and 35° C tripled and
quadrupled metabolic rate, respectively, but the resulting heat generated by
such increases accounted for increases in body temperature of less than
1°C. This was mainly caused by the fact that the increased heat production
was accompanied by increases in thermal conductance attributed to the greater
ventilatory rates needed to support the higher rates of metabolism
(Bennett et al., 2000).
Although the same phenomenon may have prevented further increases in body
temperature in C. durissus, the magnitude of this process in
rattlesnakes most likely was smaller than that recorded in Varanus.
Reptiles are known to exhibit a relative hypoventilation during digestion
(Wang et al., 2001), but while
the air convection requirement for O2 in Python was
reduced by 46% (Secor et al.,
2000), in Varanus this reduction was only 21.4%
(Hicks et al., 2000). Thus,
the heat loss due to the changes in conductance associated with the increased
total ventilatory rates during digestion should have been greater for
Varanus compared to C. durrisus. Finally, the larger
thermogenic effect of feeding in rattlesnakes compared to Varanus may
also be related to the larger metabolic response to feeding in C.
durissus; metabolism increases from 4- to 7-fold
(Andrade et al., 1997),
compared to a 3- to 4-fold change seen in Varanus
(Bennett et al., 2000).

In brooding pythons Python molurus body temperature can increase
up to 7.3°C above ambient temperature by endogenous heat production, due
to increased metabolic rates associated with the spasmodic contractions of the
body musculature (Hutchison et al.,
1966). This figure is far more impressive than the thermogenic
effect of feeding found in rattlesnakes (present study) and in varanid lizards
(Bennett et al., 2000).
Interestingly, however, brooding pythons showing such a large increase in body
temperature experience metabolic rates that are only 9.3 times higher than
non-brooding females under the same environmental conditions
(Hutchison et al., 1966).
Thus, the discrepancy between the increase in metabolism and body temperature
among brooding pythons and digesting rattlesnakes and lizards indicates that
other factors may affect the thermoregulatory ability of brooding pythons. One
likely factor is posture; by remaining coiled around the eggs, brooding
pythons decrease the surface area, which otherwise would serve as an avenue
for heat loss (see Vinegar et al.,
1970). Other possibilities are changes in conductance associated
with circulatory adjustments, however, changes in heat transport via
the circulatory system remain to be investigated.

Concluding remarks

Endotherms may use SDA or exercise-generated heat for thermogenesis, saving
a substantial amount of energy that would otherwise be used for this purpose
(Costa and Kooyman, 1984). For
an ectotherm, the general notion is that the heat generated during digestion
is a wasteful byproduct generated from the metabolic increment
(Hailey and Davies, 1987)
since they naturally do not use metabolism to generate heat for
thermoregulation. However, thermogenesis in snakes may act in concert with the
behavioral post-prandial thermophilic response to achieve the suite of
ecological and energetic benefits of increased body temperature during
digestion. Particularly poignant in the case of snakes is the long, protracted
digestion process. So, although the magnitude of the thermal increment
following feeding may seem negligible, the duration of this sustained increase
in body temperature is sufficient to suggest that digestion-derived heat in
this ectotherm is a physiologically and ecologically important phenomenon.

ACKNOWLEDGEMENTS

D.V.A. and S.P.B. were supported by Jovem Pesquisador and MSc FAPESP
grants, respectively. A.S.A. was supported by a CNPq grant. G.J.T. and W.K.M.
were both supported by operating grants from the NSERC of Canada. Partial
results of this study were presented by D.V.A. at the 2003 Joint Meeting of
Ichthyologists and Herpetologists held in Manaus, AM, Brazil, from June 26 to
July 1, 2003. D.V.A.'s participation in this meeting was sponsored by FAPESP
and FUNDUNESP.